This invention relates to the determination of the terminal sialic acid residues in human transferrin. One embodiment of the invention relates to the quantification of terminal sialic acid residues in transferrin contained in samples of human body fluids, and in particular to an interpretation of changes in the chemical structure of transferrin such as are caused, for example, by regular and excessive alcohol consumption.
There are a number of indicators used in medicine to diagnose alcohol abuse or addiction. With the help of so-called xe2x80x9cstate markersxe2x80x9d, it is possible to estimate alcohol consumption, different markers serving to estimate consumption over a short period of time (eg, blood alcohol level) or over longer periods (in particular various hepatic enzymes). A detailed review of alcoholism and suitable markers is contained in M. Soyka (publisher, Biological Markers for Alcoholism, Chapman and Hall, (1995)).
An abnormal transferrin variant has proved to be a suitable marker with a relatively high degree of specificity. The scientific studies published so far have shown that this molecular modification takes the form of a lack or a reduction in the number of sialic acid residues in the transferrin molecule.
Human serum transferrin is a glycoprotein with a relative molecular weight of about 77,000 g/mol. It consists of a single peptide chain of 679 amino acids, to which two oligosaccharide chains consisting of N-acetyl glucosamine, mannose and galactose are attached. These carbohydrate chains have two to three antennae, to the ultimate unit of which (galactose) a sialic acid residue is bound.
Regular alcohol abuse impairs the mechanism by which sialic acid is transferred to transferrin, and as a consequence there are increased numbers of transferrin isomers with fewer than the usual four-to-six terminally bound sialic acid residues (desialicized transferrin, sialic-acid deficient transferrin, sialic deficient transferrin, SDT)
According to Jong et al. (1980), the most common tetrasialotransferrin isomer (FIG. 1) is made up of:
4 terminal N-acetyl sialic acid residues, 4 galactose residues, 8 N-acetyl glucosamine residues and 6 mannose residues in each oligosaccharide side-chain of the transferrin molecule.
Methods described in the prior art are based mainly on the separation of differently glycosylated transferrin derivatives by way of their charge, eg, by way of isoelectric focusing or chromatographic techniques. Familiar methods include:
1. The isoelectric focusing method Electrophoretic separation of isotransferrins according to their isoelectric points (Pl 6.1-5.1). (Helena Stibler and Stefan Borg, Pharmacol. Biochem. Behav. 1980, 13, Suppl. 1, 47-51).
2. The chromatographic method by means of ion exchanger (Helena Stibler et al., Clinical and Experimental Research Sept./Oct. 1986, Vol. 10, No. Pages 535-543).
3. The HPLC method Saturation of the transferrin with an iron salt, followed by extraction on a column and subsequent densitometric determination. (Strahler et al., J. of Chromatography 266, 1983, 281-289).
4. Isocratic HPLC method Based on the separation of isotransferrins by means of cationic buffers. (Joustra Marius et al., Patent No. EP 0 172 217 B1), (HPLC techniques according to Jan Jeppson et al., Clin. Chemistry 39/10; 2115-2120 (1993).
5. Turbidimetric method of the company AXIS Likewise based on saturation of the isotransferrin by means of iron salt or a heterogeneous immunoassay with separation on a column followed by a turbidimetric measurement. (Patent: WO 99119983 A 911226).
6. The immunoenzymatic EIA method involving a Pharmacia conjugation Uses a monoclonal antibody after saturation of the isotransferrin with an iron salt and column separation (O. Mxc3xa4rtenson et al.).
7. Pharmacia""s RIA method: A chromatographic method using an ion exchanger after saturation of the isotransferrin with an iron salt and quantitative determination by means of radio-immunoassay.
A The above-mentioned methods, however, are not yet satisfactory for the user. The likelihood of false diagnoses and the very complicated and time-consuming test procedures are particularly problematical.
The prior art also describes a class of proteins or glycoproteins referred to as lectins, which bind to certain carbohydrate configurations and to glycoproteins which carry such carbohydrate configurations. On account of their specificity for certain carbohydrates, such lectins have been used, eg, for the blood-group-specific agglutination of erythrocytes.
The object of this invention was to provide a method allowing rapid and uncomplicated determination of terminal sialic acid residues in human transferrin.
This object is established according to the invention by a method of determining terminal sialic acid residues in human transferrin contained in a sample fluid, said method being based on a sandwich principle and being characterized in that the sample fluid is incubated with a first receptor which binds specifically to human transferrin, the thus-formed complex is separated from the sample fluid and incubated with a second receptor which binds specifically to terminal sialic acid residues in human transferrin, the second receptor being bound to a marker or having the ability to bind thereto, and the complex comprising the first receptor, human transferrin and the second receptor is determined by means of the marker.
It is generally preferable, in keeping with familiar and time-tested technology, to use an anti-transferrin antibody as the first receptor. It is of advantage if the first receptor is a polyclonal anti-transferrin antibody. Antibodies of this kind are commercially available (eg, Sigma, No. T2027). However, there are other receptors specific to human transferrin which are suitable too, provided they do not significantly impair the binding of the second receptor to the sialic acid residues. It would be conceivable, for example, to use human transferrin receptor.
The second receptor is preferably a lectin, the most preferred of which is the lectin Sambucus nigra; its production has been described by Brokert et al. (Biochem. J. 221, 103-109 (1984)).
According to one embodiment of the method of the invention, the first receptor is bound to a solid phase, as a result of which, during incubation, the transferrin becomes attached to the solid phase. According to another embodiment, however, the first receptor can also be present in solution; in this case the receptor is able to bind to a solid phase by means of a specific pair of binding agents, one of which is bound to the solid phase and the other to the receptor. Suitable pairs of binding agents are known to those versed in the art.
It is expedient to use a wall of a reaction vessel, such as a sample tube, a microplate or a cuvette as solid phase. The solid phase can also consist of a particulate material such as polystyrene or magnetic beads.
In the same way, the second receptor can either be bound directly to the marker or else be capable of binding thereto by way of a specific pair of binding agents. According to a preferred embodiment, the specific binding agents are biotin and streptavidin or avidin, with the second receptor typically carrying the biotin and the marker the streptavidin/avidin.
The sample fluid is incubated with the first receptor and the second receptor for a period of 10 to 60 minutes in each case, preferably 20 to 40 minutes, and at a temperature of 10 to 40xc2x0 C. A 30-minute incubation period at room temperature provides good results.
Basically, all detection techniques commonly used in connection with immunoassays are suitable for determining the marker on the second receptor. Persons versed in the art are familiar with such techniques, and they do not need to be explained in detail here. They include, in particular, enzymatic techniques, ie, techniques in which the marker is an enzyme which is quantified by determining a substrate. Enzymes commonly used for such purposes include, eg, phosphatases such as alkaline phosphatase, oxidases, peroxidases, dehydrogenases such as glucose-6-deghydrogenase, hydrolases such as urease, etc. The enzymatic reaction typically generates a chromogenic, luminescent or fluorescent substrate, which can then be determined using a suitable method, usually a photometric method. Suitable chromogens include, eg, ABTS (2,2xe2x80x2-azino-bis-(3-ethyl)-benzthiazolin-6-sulfonic acid), orthophenyldiamine, tetramethylbenzidene, 5-amino-salicyclic acid, etc. Enzymatic techniques also encompass, eg, modifications to chemical or physical parametersxe2x80x94such as light absorptionxe2x80x94which are caused by the substrate reaction. This means that the sialic acid residues can also be determined by UV methods with alcohol dehydrogenase, glucose-6-dehydrogenase etc, and by means of the coenzyme AND, NADP etc, or by using a diaphorase in the presence of NADP and the chromogen iodonitrotetrazolium violet in order to obtain a colored formazan derivative.
Besides enzyme markers, there are naturally other methods of marking the second receptor, eg, through use of fluorescent or luminescent tracers, or of radiotracers; radioactive labelling, however, is not recommended for practical reasons. Immunofluorescence tests can be carried out, eg, using a conjugate of the second receptor with IFTC (fluorescin isothiocyanate).
As sample fluid containing the transferrin to be determined, use is generally made of a body fluid such as blood, serum, urine, liquor, vitreous humor, bile, abdominal fluid, etc. If necessary, use is made of a body fluid which has been modified by chemical or physical treatment but which, however, is preferably free of anticoagulants. It is preferable if whole blood or serum is used as body fluid.
The subject matter of the invention also includes a method of determining sialicacid-deficient transferrin in body fluids, characterized in that
(a) A sample of body fluid is assayed using a method according to one of the preceding claims,
(b) the terminal sialic acid residues are determined for at least one standard which contains a defined quantity of a substance comprising sialic acid residues capable of binding to the second receptor, and
(c) the content of sialic-acid-deficient transferrin is ascertained on the basis of the results obtained in step (a) and step (b).
It is preferable if step (b), ie, determination of sialic acid residues using known formulations, is repeated several times, each time with the standard containing a different quantity of sialic acid residues capable of binding to the second receptor. It is especially beneficial if three to seven, most preferably five to six measurements are carried out on different standards, and a calibration curve plotted from the results obtained. By comparing the readings obtained for the sample fluid with the calibration curve, the content of sialic-acid-deficient transferrin can be determined with a high level of accuracy.
As is obvious to a person versed in the art, it is a good idea to determine suitable negative and/or positive reference samples at the same time.
According to one embodiment, a standard is used which contains transferrin with a defined quantity of sialic acid residues. By this, one understands body fluids, especially pooled body fluids, and suitably formulated compositions. Suitable concentrations of sialic acid residues in the standards used to plot the calibration curve range from 0 to 50 xcexcmol/dl, preferably from 2.5 to 25 xcexcmol/dl.
In another embodiment, the standard contains a substance other than transferrin, with a defined quantity of sialic acid residues capable of binding to the second receptor. Examples of such substances are immobilized mucin and/or an immobilized oligosaccharide with a defined quantity of sialic acid residues capable of binding to the second receptor, eg, a mucin which is obtainable from Sigma (Sigma No. M 3895, mucin from bovine submaxillary glands). In this specification, the terms xe2x80x9cimmobilizedxe2x80x9d and xe2x80x9cimmobilizablexe2x80x9d are used in the sense of xe2x80x9cbeing bound toxe2x80x9d and xe2x80x9cbeing capable of binding toxe2x80x9d the solid phase.
Another example is N-acetylneuramine-lacto-N-neo-tetraose c (xcex1Neu 5 AC-(2-6)-xcex2Gal-(1-4)-xcex2GlcNAc-(1-3)-xcex2Gal-(1-4)-Glc (Sigma No. A4814) combined with a solid phase coated with concavalin A (Sigma No. C7275) (specific for xcex1-D-glycosyl bonds).
In cases where a fluid containing transferrin is used as standard, the transferrin is attached to the solid phase by means of the first receptor. Where a standard that contains a substance other than transferrin is used, it is not possible to attach the marker by means of a complex comprising the first receptor and transferrin, as will be obvious to a person versed in the art. In this case, the substance containing the sialic acid residues is accordingly immobilized, ie, is bound to a solid phase or is capable of binding thereto.
The method of the invention can be used to determine SDT in samples of body fluids from male and female donors. In the literature, healthy persons are reported as having a physiological transferrin content of 260 mg/dlxc2x115%. Surprisingly, the method of the invention also provides accurate results for pregnant women.
According to an additional aspect, the invention also relates to the use of a lectin, especially the lectin Sambucus nigra, in a process for determining sialic-acid deficient transferrin.
Since sialic-acid deficient transferrin is a reliable marker for alcoholism, as was mentioned at the beginning, the method of the invention can considerably facilitate the diagnosis of alcohol abuse.
The subject matter of the invention thus also includes a method, eg, use of the aforementioned process, of diagnosing alcohol abuse or addiction. To this end, sialic-acid deficient transferrin is determined in a sample of body fluid and then compared with normal levels to see whether there is a pathological discrepancy in the number of terminal sialic acid residues. Comparison is carried out in the usual way, eg, using reference samples taken from patients and/or healthy persons. Alternatively, or in addition, it is also possible to calculate whether the measured level is within the normal range or not.
Further applications in this connection include, eg, the early recognition of regular alcohol abuse and alcoholism, monitoring therapeutic treatment of withdrawal patients, use as a differential-diagnostic instrument to distinguish between diseases induced by alcohol (eg. liver) and those of non-alcoholic origin, a laboratory-diagnostic aid in deciding on the start of pharmacoprophylaxis in intensive medicine, and generally in occupational and forensic medicine, eg, for providing expert opinions.
According to yet another aspect, the invention also relates to a reagents kit suitable for determining the terminal sialic acid residues in transferrin. A kit of this kind comprises an immobilized first receptor which binds specifically to human transferrin and, physically separated therefrom, a second receptor which carries a marker or is capable of binding to a marker and which binds specifically to terminal sialic acid residues in human transferrin, and also, if necessary, a solid phase which is capable of binding specifically to the first receptor, a marker which is capable of binding specifically to the second receptor, a substrate for the marker, and the customary adjuvants, buffers and additives.
The first receptor is preferably an anti-transferrin antibody, in particular a polyclonal anti-transferrin antibody, and the second receptor is preferably a lectin, in particular the lectin Sambuca nigra. 
The reagents kit may contain, in addition, at least one standard which contains a defined quantity of a substance comprising sialic acid residues capable of binding to the second receptor, as was described before. This substance is preferably N-acetyineuramine-lacto-N-neo-tetraose c (xcex1Neu 5 AC-(2-6)-xcex2Gal-(1-4)-xcex2GlcNAc-(1-3)-xcex2Gal-(1-4)-Glc (Sigma No. A4814) which is capable of binding to a solid phase coated with concavalin A (Sigma No. C7275), or a mucin with a defined quantity of xcex1-anomeric (2-6) sialic acid residues (eg, Sigma No. M3895, mucin from bovine submaxillary glands).
Yet another part of this invention""s subject matter is a test strip for the qualitative and semi-quantitative determination of SDT, comprising
(a) a sample application zone,
(b) at least one reaction zone, containing a first receptor which can be immobilized on a solid phase and which binds specifically to human transferrin, and a marked second receptor which binds specifically to terminal sialic acid residues in human transferrin, and
(c) a detection zone for immobilizing the first receptor, said zone containing a substance which binds specifically to said first receptor.
Test strips of this sort are known to persons skilled in the art, making a detailed description unnecessary. Briefly, a strip of this kind consists of a material through which a sample fluid containing a substance to be determined can migrate, eg, by capillary force or gravity, and which is inert with respect to the substance to be determined. After the fluid has been applied to the sample application zone, it migrates through a reaction zone, with ternary complexes of the first receptor, transferrin and the second, marked receptor being formed. These are subsequently immobilized in the detection zone. Non-bound reactants and substances migrate right through the detection zone and are collected in an end zone.
The first receptor is preferably an anti-transferrin antibody, in particular a polyclonal anti-transferrin antibody. The first receptor is preferably immobilized by means of biotin and streptavidin/avidin. The second receptor is preferably a lectin, in particular the lectin Sambucus nigra. Markers for the second receptor which are suitable for use in such test strips are known to persons versed in the art and include, eg, gold and silver markers and chromogens.
According to a particularly preferred embodiment, the test strip also includes at least one reference zone for negative and/or positive referencing. A negative referencing zone corresponds for the most part with the detection zone, except that there is no binding partner to immobilize the first receptor. A positive reference zone expediently contains a defined quantity of immobilized sialic acid residues which are capable of binding to the second receptor. For a semiquantitative assay, a number of positive reference zones can be provided which have different amounts of sialic acid residues. Other test strip variants and layouts are described in the prior art.
The method of the invention can also be used to determine terminal sialic acid residues in all natural proteins which have siatic acid residues bound in the 2-6 position to xcex2-galactose and N-acetyl glucosamine.
The method of the invention is explained in more detail by means of the following examples, together with FIGS. 1 and 2.